Moderately thermostable GH1 β-glucosidases from hyperacidophilic archaeon Cuniculiplasma divulgatum S5

Abstract Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus, and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5–6.0 (CIB12) and 4.5–5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma.


Introduction
Extremophiles thriving in environments with physico-chemical conditions hostile to common micr oor ganisms, ar e widel y distributed across the globe.Archaea often outcompete bacteria and eukaryotes in extreme en vironments , and flourish at high temper atur es, at low or high pH values and at ele v ated salinities (Shu and Huang 2022 ).They employ various and often unique physiological pr operties, whic h r esult in pr oducts of biotec hnological importance useful for applications in different industries (Elleuche et al. 2014 ).One of suc h or ganisms is Cuniculiplasma divilgatum , ubiquitous in moderate-to-low temperature acid mine drainage systems, with pH optimum at 1.0-1.2(Golyshina et al. 2016a ,b ).Cuniculiplasma spp.are also found in geothermal areas worldwide, pointing at their global distribution in acidic environments of different origin and at variety of temperature adaptations in these arc haea (Gol yshina et al. 2019 ).Taxonomicall y, the genus Cuniculiplasma is included in the order Thermoplasmatales , which contains organisms with the lo w est pH values for growth ever recorded (Golyshina et al. 2019 ).Potentially, Cuniculiplasma spp.can serve as a source of extremozymes, enhancing our comprehension of these enzymes' functions and their significance in the life strategies and ecology of extremophilic archaea, while also offering promise for potential biotechnological uses.
Cuniculiplasma divulgatum S5 genome encodes 22 glycoside hydrolases (GHs), according to CAZY records ( http://www.cazy.org/a7595.html; Cantar el et al. 2009 ), thr ee of whic h belong to the Gl ycosyl Hydr olases Famil y 1 (GH1) that encompasses a large set of enzymes sharing a catalytic domain with a ( α/ β)8 TIMbarrel fold and a retaining mechanism of catalysis with activities spread across 34 EC numbers (CAZY database; Cantarel et al. 2009 ).These enzymes are ubiquitous across all domains of life and play fundamental r oles, suc h as in vivo degradation of lignocellulosic materials for n utrient uptak e.Ad ditionally, the y have a wide range of in vitro applications in food, medicine, and the production of bio-based chemicals and renewable energy sources (Cantarel et al. 2009 , Ketudat Cairn andEsen 2010 ).GH1 enzymes are encoded in genomes of archaea, with 25 enzymes functionall y c har acterized, including fr om two taxonomic neighbours of C. divulgatum , Thermoplasma acidophilum DSM 1728 (Kim et al. 2009 ), and from Picrophilus torridus DSM 9790 (Murphy and Walsh 2019 ) .Cuniculiplasma divulgatum encodes three such GH1 proteins, one of which was partially characterized, ho w ever, only with model, p -nitr ophen yl ( p NP) glucoside substrate, with no activity determined against natural substrates (He et al. 2023 ).
Cuniculiplasma spp.are known (Golyshina et al. 2016a ,b ), like their closest r elativ es fr om Thermoplasmatales (Huber and Stetter 2006 ), to r el y on scav enging detritus/dead cell biomass of micr oor ganisms, and corr espondingl y, their gr owth media contain complex organic peptide-containing ingredients, such as yeast, meat, or beef extract, tryptone, or peptone .T hey occupy niches with other related Thermoplasmatales , but also, intriguingly, with primary producing, photosynthetic organisms, such as Chlamydomonas acidophila or Euglena mutabilis (Distaso et al. 2022 ) that suppl y pol ysacc harides in the envir onment.It is known that monomeric sugars did not enhance growth of C. divulgatum, howe v er, the ability of Cuniculiplasma spp. to make use of other products/intermediates of pol ysacc haride hydr ol ysis, r emains to be assessed.
The aim of present study was therefore to heterologously expr ess, purify, and structur all y and functionall y c har acterize, the GH1 carbohydr ate-activ e enzymes fr om C. divulgatum.Mor eov er, to assess the ecological importance of these enzymes for Cuniciliplasma spp. in their natural habitats, we examined the enzyme substrates for their ability to support the growth of these microorganisms.
In this w ork, w e report the results of our study on two βglucosidases CIB12 and CIB13 with respect to their phylogeny, bioc hemical pr operties, and substr ate specificities with model and natur al substr ates, accompanied with the anal ysis of their r esolv ed crystal structur es .T hese intracellular enzymes ha ve highest activities to w ar ds cellobiose, amendment of which to the medium significantl y incr eased the biomass yield of C. divulgatum, pointing at physiological and ecological importance of these GH1 famil y cellobiohydr olases .
As the primary carbon source, the beef extract (ThermoFisher Scientific, Paisle y, UK) was ad ded at final concentration 3 g l −1 .In addition, cellobiose and lactose (Sigma-Aldrich and Scientific Laboratory Supplies, Nottingham, UK, respectively) were tested as carbon sources at concentration of 3 mM (0.1% w/v).The pH of the medium was adjusted to 1.0 with concentrated H 2 SO 4 .Cultivation was conducted at 37 • C in Erlenmeyer flasks in an orbitary shaker at 100 r m −1 .The growth was followed by measuring the culture optical density at 600 nm using a BioPhotometer Plus (Eppendorf, Hambur g, German y).Consumption of added lactose and cellobiose during the growth of C. divulgatum was measured by HPLC anal ysis of cultur e supernatants using a Shimadzu Pr ominence-I LC-2030c 3D Plus instrument.Samples of growth media were centrifuged at r elativ e centrifugal force (rcf) of 10 000 for 10 min, filter ed thr ough n ylon filters (0.22 μm), and 10 μl-aliquots of cleared samples were used for HPLC analysis.Cellobiose was analysed using a Bio-Rad Aminex 87H columns at 50 • C (mobile phase 5 mN H 2 SO 4 , 0.6 ml min −1 ), whereas lactose was measured using an Aminex 87P column at 80 • C (mobile phase H 2 O, 1 ml min −1 ).
Esc heric hia coli str ains used in this study wer e cultiv ated on Luria-Bertani (LB) medium (per litre of deionised water, 10 g tryptone, 5 g yeast extract, and 5 g NaCl) with 15 g l −1 agar amended in solid media, at 37 • C. Strains harbouring the recombinant plasmids were grown in LB medium with ampicillin at the final concentration of 100 μg ml −1 (Li et al. 2021 ).

GH assays with chromogenic and na tur al substr a tes
GH activity of purified GH1 proteins w as assay ed using tw o panels of c hr omogenic (model) and natural GH substrates ( Tables S1  and S2 ).The r eaction mixtur es contained 20 mM MES buffer (pH 5.0), 1 mM p NP-gl ycosyl substr ate, and 3 μg of enzyme in the final volume of 200 μl (incubation at 37 • C for 120 min).The absorbance of the released p -nitrophenol was measured at 410 nm.One unit (U) of enzyme activity was defined as the amount of enzyme needed to liberate 1 μmol of p NP per minute under the assa y conditions .T he specific activity of enzyme r eferr ed to the number of units of enzyme activity per mg of protein.Reaction mixtures for assays with natural substrates (0.2 ml) contained 20 mM MES buffer (pH 6.0), 1 mM substrate, and 3 μg of purified enzyme .After o vernight incubation at 30 • C, 10-μl aliquots of reaction mixtures were used to determine the concentration of released reducing sugars using a modified bicinchoninic acid (BCA) assa y (Millipore , Gillingham, UK) by adding 10 μl of 2 M NaOH and 200 μl of fresh BCA reagent mix.After 30 min incubation at 60 • C in a shaker (500 r m −1 ), the solution absorbance was measured at 562 nm.A standard curve was pr epar ed by plotting the avera ge blank-measur ement for eac h r educing sugar monomer concentration used to calculate the released monomer concentration in reactions (Li et al. 2021 ).GH activity of purified wild-type and mutant enzymes was determined under indicated reaction conditions using 2 mM p NP-βd -glucopyranoside or 25 mM cellobiose as substrates.All assays were performed in triplicate, and the results are presented as means ± SD (standard deviations) from at least two independent experiments.One unit (U) of enzyme activity was defined as the amount of enzyme r equir ed to pr oduce 1 μmol of p NP per minute under the assay conditions.

Kinetic parameters
Michaelis-Menten constant ( K M ) and maximum reaction velocity ( V max ) of purified CIB12 and CIB13 were determined by assaying enzymes activity in the presence of increasing concentrations of substr ates: p NP-βd -glucopyr anoside (0-10 mM), cellobiose or lactose (0-150 mM) in 50 mM MES buffer (pH 5.0) using 3 μg or 5 μg of enzyme, r espectiv el y, per 0.2 ml reaction.For assays with p NPβd -glucopyr anoside as substr ate (30 min incubation at 30 • C), the absorbance of released p NP was measured at 410 nm after adding 10 μl of 1 M Tris-Cl buffer (pH 9.0) for complete colour de v elopment.For assays with natural substrates, the reaction mixtures were incubated at 30 • C for 4 h and then filtered using centrifugal filters (PES, 10 kDa cut-off, VWR, 10 000 × g ). 10 μl aliquots of filtr ate wer e used for the HPLC anal ysis of r eleased glucose using an Aminex 87H column (60 • C, mobile phase 5 mN H 2 SO 4 , isocratic flow at 0.6 ml min −1 , RI detector).The standard curve was used to determine glucose concentrations in samples and then calculate enzyme activity ( μmol min −1 mg −1 ).The obtained values were fitted to the classical Michaelis-Menten equation, and the values for V max , k cat , K M , and catalytic efficiency ( k cat / K M ) were calculated using a Gr a phP ad Prism software (Swift 1997, Li et al. 2021 ).

Analysis of optimal reaction conditions (pH, temper a ture, NaCl, and Tween-20)
The pH dependency of GH activities of CIB12 and CIB13 was investigated by incubating the purified enzymes (3 μg per 0.2 ml reaction mixture) in 20 mM Britton-Robinson pH buffer (pH 3.0-12.0)at 30 • C for 120 min with 2 mM p NP-βd -glucopyranoside as substrate.After incubation, 10-μl aliquots of 1 M Tris-HCl buffer (pH 9.0) were added to each assay for complete colour de v elopment and pr oduced p -nitr ophenol ( p NP) was determined spectr ophotometrically at 410 nm.Temperature profiles of GH activity of purified CIB12 and CIB13 with 2 mM p NP-βd -glucopyranoside as substr ate wer e measur ed at temper atur es 25 • C-90 • C using 3 μg of protein per reaction in 50 mM MES buffer (pH 5.0).Reaction mixtur es wer e incubated at indicated temper atur es for 120 min, and the absorbance of the released p NP was measured at 410 nm.The effect of NaCl concentrations (0-4.0M) on enzymatic activity of CIB12 and CIB13 (3 μg of protein per reaction) was determined using 25 mM cellobiose as substrate (due to high backgr ound observ ed with p NP-βd -glucopyr anoside in the pr esence of high NaCl concentrations).Reaction mixtures (0.2 ml) contained 50 mM MES buffer (pH 5.0), 25 mM cellobiose, and 3 μg of protein (incubation for 240 min at 30 • C).The effect of Tween-20 (a detergent) on enzymatic activity of CIB12 and CIB13 was investigated using r eaction mixtur es (0.2 ml) containing 50 mM MES buffer (pH 5.0), 1 mM p NP-βd -glucopyranoside, and 3 μg of protein (incubation for 120 min at 30 • C).

Analysis of enzyme thermostability and dena tur a tion temper a ture
Purified pr oteins wer e incubated at differ ent temper atur es r anging from 30 • C to 80 • C for 1-5 h, and the residual enzyme activity was measured at 30 • C using p NP-βd -glucopyranoside as substrate (8.9 mM for CIB12 and 1.8 mM for CIB13) in 20 mM HEPES buffer (pH 7.0) for CIB12 or in 20 mM MES buffer (pH 6.0) for CIB13, 3 μg of enzyme in 200 μl r eaction mixtur es.Pr otein melting temper atur es of purified enzymes wer e determined using differential scanning fluorimetry (DSF) with Sypro Orange ® (Invitrogen, T hermo Fisher Scientific , USA) as a re porter d ye (Elgert et al. 2020 ).Samples w ere loaded into 96-w ell opticall y tr ansparent plates (Bio-Rad, USA), and the heating rate was 1.0 • C min −1 with fluor escence r eadings (excitation at 530 ± 30 nm, emission at 575 ± 20 nm) taken after each 1 • increase .T he reaction mixtures containing 10 μg of enzyme, 25 X Sypro Orange ® dye and 20 mM buffer (for CIB12: HEPES buffer, pH 7.0; CIB13: MES buffer, pH 6.0) were mixed and heated from 25 • C-95 • C in increments of 1 • C min −1 on a CFX96 Real-Time System-C1000 thermal cycler (Bio-Rad).All measur ements wer e done in triplicate.SYPRO Orange dye interacts with a protein undergoing thermal unfolding, with its fluorescence increasing upon exposure to the protein's hydr ophobic cor e .T he T m for CIB12 and CIB13 were determined as the melting temper atur e that correlates with half of the maximal fluor escence signal fr om data plotted using Boltzmann equation in Gr a ph P ad Prizm 6.0.
Diffraction data was collected at 100 K on a Rigaku home source Micromax-007 with R-AXIS IV ++ detector and processed using HKL3000 (Minor et al. 2006 ).Both CIB12 and CIB13 structures wer e solv ed by molecular replacement using Phenix.phaser(Liebschner et al. 2019 ), and models were generated by the Phyre2 serv er (K elley et al. 2015 ) onto the structur e of a β-glucosidase from Pyrococcus furiosus (PDB code 3APG).Model building and refinement were performed using Phenix.refineand Coot (Emsley and Cowtan 2004 ).Tr anslation/Libr ation/Scr e w (TLS) par ameterization was utilized for refinement and B -factors wer e r efined as isotr opic.Structur e geometry and v alidation wer e performed Figure 1.A Neighbour-joining phylogenetic tree of CIB12 and CIB13 with their functionally and/or structurally characterized GH1 family homologues.Sequences were aligned using Muscle (Edgar 2004 ), bootstrap values were calculated with 1000 pseudoreplicate trees , P oisson substitution model was used.Evolutionary analyses were conducted in MEGAX (Kumar et al. 2018 ).Scale bar, 0.2 substitutions per position.
using Phenix Molprobity tools.Data collection and refinement statistics for both structur es ar e summarized in Table S3 .The structur es wer e deposited to the Protein Data Bank with PDB accession codes 8U7F (CIB12) and 8U7G (CIB13).
The phylogenetic tree was built using neighbour-joining method (Saitou and Nei 1987 ), evolutionary distances were computed using the Poisson correction method (Zuckerkandl and P auling 1965 ), for bootstr a p anal ysis, 1000 tr ee pseudor eplicates were used (Felsenstein 1985 ), all of which are a part of MEGA X pac ka ge (Kumar et al. 2018 ).3D protein structures were analysed using Chimera X v.1.4and PyMOL (the PyMOL Molecular Gr a phics System, version 3.0 Schrodinger, LLC).

CIB12 and CIB13 cocluster with homologues from Thermoplasmatota and Thermococcota
Curr entl y, 186 families of glycosyl hydrolases are classified, and they ar e gr ouped into 16 differ ent superfamilies and added clans (Shriv astav a 2020 ).To classify the CIB12 and CIB13, they were scr eened a gainst InterPr o database and identified as GH1 famil y of enzymes.Both enzymes belong to IPR017853 superfamily of glycosyl hydr olases, mor e specificall y to GH1 famil y, r epr esented in InterPro database with IPR001360 family.In Pfam database these enzymes belong to PF00232 domain proteins, with protein fingerprints PR00131 in PRINTS database.Glycosyl hydrolases in the GH1 family are ubiquitously distributed across the tree of life, appearing in both eukaryotic and prokaryotic organisms due to their div erse substr ate specificities.In IPR001360 family, total number of species are 16 034, where bacteria account for 13 216 r epr esentatives, eukaryotic species for 2544 and archaeal species for 250 ( https:// www.ebi.ac.uk/ inter pr o/ entry/ InterPro/ IPR001360 ).To establish the phylogenetic affiliation of CIB12 and CIB13 with characterized gl ycosyl hydr olases, initiall y, 260 sequences r efer enced as "r e vie wed" in the UniPr ot database wer e used (UniPr ot Consortium 2019 ).The majority of br anc hes within the tree encompass sequences of eukaryotic glycosyl hydrolases sourced from Homo sapiens as well as model plants Arabidopsis thaliana and Oryza sativa ( Fig. S1 A).Interestingly, two distinct branches have only prokaryotic sequences of GH1 family, in contrast to five branches having solely eukaryotic sequences, though as pr e viousl y mentioned, eukaryotic enzymes are less presented, but more studied.Figure 1   47%) (Cubellis et al. 1990 ), and β-glucosidase CelB from P. furiosus (44% and 46%) (Li et al. 2013 ), β-glucosidase BglB from Thermosphaera aggregans (46% and 46%) (Chi et al. 1999 ) and Thermococcus kodakarensis KOD1 (38% and 40%) (Hwa et al. 2015 ).Amino acid sequence alignment suggested that Glu204 and Glu388 represent the catalytic glutamate residues of CIB12, whereas Glu204 and Glu385 are present in CIB13.As expected, these amino acid r esidues ar e highl y conserv ed in all GH1 members.

CIB12 and CIB13 are GH1 GHs with a preference towards cellobiose
In this study, CIB12 and CIB13 were recombinantly expressed and purified to homogeneity by Ni-affinity c hr omatogr a phy as described in the section "Materials and methods".SDS-PAGE analysis of purified protein samples revealed the presence of one major band corresponding to CIB12 and CIB13 ( Fig. S2 ).The apparent molecular masses of purified recombinant proteins were estimated to be ∼56 kDa, whic h ar e close to the expected protein sizes.
According to the CAZy and BRENDA databases, bioc hemicall y c har acterized GH1 GHs were active against a broad range of natural and chromogenic ( para -nitrophenyl, p NP) substrates, such as p NP-βd -glucopyranoside and p NP-βd -galactopyranoside .T herefore, purified CIB12 and CIB13 were first screened for hydr ol ytic activity against a panel of 21 c hr omogenic p NPsubstrates (Fig. 2

Optimal pH for GH activity of CIB12 and CIB13 is w eakl y acidic
pH-dependency of CIB12 activity was investigated in the range of pH from 3.0 to 12.0.CIB12 was found to be activ e ov er a br oad pH r ange fr om 4.0 to 8.5 (Fig. 4 A).The enzyme activity was maximal between pH 4.5 and 6 and was > 50% at pH 4.0 and 6.5.pH-dependency of CIB13 was also investigated in the same pH r ange, 3.0-12.0.CIB13 was activ e in a narr o w er pH range.As shown in Fig. 4 (B), the optimal pH (pH opt ) of CIB13 was at 5.0, and it retained at least 75% activity at pH 4.5 and 5.5, which is consistent with other c har acterized β-glucosidases exhibiting their highest activities within the acidic pH r ange, typicall y between pH 4 and 6.5 and having lo w er activity and may become unstable when exposed to mildly alkaline conditions.CIB12 and CIB13 exhibited pH opt for activities within the same range as their characterized counter parts fr om v arious thermoacidophilic arc haea: P. furiosus (Voorhorst et al. 1995 ) with a pHopt of 5.5, P. torridus with a pH opt of 5.0-5.5 (Murphy and Walsh 2019 ), S. solfataricus P2 with a pH opt of 5.5, and T. acidophilum with a pH opt of 6.0 (Kim et al. 2009 ).CIB12 and CIB13 ar e deriv ed fr om the hyper acidophilic organism C. divulgatum S5, which optimally grows at pH 1.0-1.2(Golyshina et al. 2016b ).Ho w ever, both enzymes have a low probability of containing signal peptides, suggesting that they are likely intr acellular (nonsecr eted) pr oteins.In that context, note worthy ar e earlier r ecords on r educed cytoplasmic pH v alues (4.6) in

Optimal temper a tures for GH1 activities of CIB12 and CIB13 are higher than those in situ
Based on the results with the pr eferr ed model substr ate p NP-βd -glucopyranoside CIB12 exhibited high activities in the temperatur e r ange 30 • C-60 • C, whic h wer e all within 75% of its maxim um activity at 50 • C (Fig. 4 C).The activity shar pl y decr eased abov e 60 • C, and no enzyme activity was observed at temperatures above 75 • C. Compared to CIB12, CIB13 was active in a narro w er temperatur e r ange (25 • C-60 • C) with the maximal activity observed at 50 • C (Fig. 4 D).Activity was not measurable at 70 • C and abo ve .Both enzymes sho w ed a good thermostability at 40 • C e v en after 5 h of incubation and retained 71.3% and 96.0% of activity for CIB12 and CIB13, r espectiv el y, after 1 h incubation at 50 • C, but were not very stable at 50 • C for longer > 2 h incubations (Fig. 5 A and B).Correspondingl y, enzymes wer e denatur ed upon temper atur e incr ease as indicated by DSF, with melting points at 61 • C and 64 • C for CIB12 and CIB13, r espectiv el y (Fig. 5 C and D), which is consistent with their optimal and maximal temper atur es for activity.Ob viousl y, GH1 enzymes from Cuniculiplasma did not exhibit optimal temper atur es as high as their closest homologues from T. acidophilum (90 • C) (Kim et al. 2009 ) and Picrophilus oshimae (70 • C) (Murphy and Walsh 2019 ), but it should be noted that optimal temper atur es of enzymes w ere w ell above the optimal temper atur es for gr owth of C. divulgatum S5 (37 • C-40 • C) and m uc h higher than av er a ge temper atur es in the isolation site (Golyshina et al. 2016a ).As reported earlier, the close relatedness of Cuniculiplasma spp .to thermophilic members of Thermoplasmatales , including Thermoplasma spp.and Thermogymnomonas spp. that are coclustering close to the Last Thermoplasmatales Common Ancestor root may point at a possible retention of some high-temperature-active enzymes inherited from their thermophilic ancestors (Bargiela et al. 2023 ), which could explain the discrepancies in temperature optima for enzyme activities and for growth.

Salt and solvent tolerance
CIB12 and CIB13 activities were tested in the presence of NaCl concentr ations v arying fr om 0 to 4 M. T he assa ys sho w ed high NaCl tolerance of CIB12 activity with cellobiose: the activity remained high (80%-100%) at 0-1.5 M NaCl and gr aduall y decr eased to 50% at 4 M (Fig. 4 E).CIB13 exhibited a steeper activity loss and sho w ed ca 30% activity at 2.5 M and no activity at 4 M (Fig. 4 F).Reports on salt tolerance are rare with some evidence where enzyme from GH1 shows salt tolerance such as enzyme from marine bacterium Alteromonas sp.L82 being active at 2 M NaCl (Sun et al. 2018 ) and enzyme from hyperthermophilic archaeon Thermococcus sp.retaining 100% activity across all NaCl concentrations (1-5 M) (Sinha and Datta 2016 ).Tween-20, a widely used nonionic detergent often emplo y ed for em ulsion pr epar ation, was tested at concentrations 0%-7%.For CIB12 (Fig. 4 G), it was observed that enzyme activity was high between 0% and 1% Tween-20 after which Table 1.Kinetic parameters of purified CIB12 and CIB13 with various substrates at 30 • C and optimal pH. it gr aduall y decr eased upon incr ease in Tween-20 concentr ation, and halved at 5% of Tween-20, and dropped to < 2.5% at 6% of Tween.In contrast to that, CIB13 (Fig. 4 H) enzyme activity was str ongl y pr omoted at 0.01%-1% of Tween-20 and then gr aduall y decreased as the Tween-20 percentage in the reaction mixture incr eased.At maximal concentr ation tested, 7% of Tween-20, CIB13 retained > 80% of its activity.The kinetic parameters of purified CIB12 and CIB13 were investigated using the optimal reaction conditions and p NP-βdglucopyranoside , cellobiose , and lactose as variable substrates (Table 1 ).With p NP-βd -glucopyranoside as substrate, CIB12 sho w ed the V max at 0.61 U mg −1 with K M value of 0.5 mM, whereas CIB13 exhibited a slightly higher activity (0.93 U mg −1 ) but lo w er substrate affinity ( K M 0.9 mM).Both enzymes also revealed similar catalytic efficiencies ( k cat / K M ) with p NP-ß-d -glucopyranoside, whic h wer e 1.1 mM −1 s −1 for CIB12 and 1.0 mM −1 s −1 for CIB13 (Table 1 ).With natural substrates (cellobiose and lactose), both enzymes r e v ealed a higher affinity to cellobiose ( K M 26.2 mM and 13.6 mM) compared to lactose ( K M 50.7 mM and 100.5 mM) for CIB12 and CIB13, r espectiv el y.CIB12 demonstr ated higher activity with natur al substr ates ( V max 1.6-1.7 U mg −1 ) compar ed to CIB13 ( V max 0.14-0.49U mg −1 ), but the latter r e v ealed a higher affinity to cellobiose ( K M 13.6 mM) (Table 1 ).Most of the curr entl y known archaeal GH1 β-glucosidases were characterized using chromogenic ( p NP) substrates and usually preferred p NP-ß-d -glucopyranoside and p NP-βd -galactopyranoside as substrates (Grogan 1991, Hei and Clark 1994, Ezaki et al. 1999 , Sriv astav a et al. 2019 ), while Tk βgl y fr om T. kodakarensis KOD1 had the highest catalytic efficiency ( k cat / K M ) with p NP-ß-d -fucopyranoside (Hwa et al. 2015 ).

Crystal structures of CIB12 and CIB13 reveal their acti v e sites
Purified CIB12 and CIB13 were crystallized using the sitting-drop v a por diffusion method, and their crystal structur es wer e determined to 2.55 Å and 2.22 Å r esolution, r espectiv el y, by molecular replacement ( Table S3 , see the section "Materials and methods" for details).Both structures revealed a classical ( β/ α) 8 TIM-barrel fold with a spherical sha pe, whic h is typical for GH1 enzymes and is one of the most common protein folds (Fig. 6 ) (Nagano et al. 2002 ).The inner wall of the TIM barrel includes the eight parallel β-str ands, whic h ar e surr ounded by eight α-helices (Fig. 6 ).In addition, the CIB12 structure contains two small antiparallel and one parallel β-sheets and six short α-helices, whereas CIB13 has four antiparallel β-sheets with two β-strands each and four α-helices outside the barrel structure (Fig. 6 ).Both CIB12 and CIB13 structur es r e v ealed the pr esence of two protomers per asymmetric unit forming a dimer with interacting residues located primarily on two C-terminal α-helices, one side β-strand, and connecting loops ( Figs S4 and S5 ).Accordingl y, the r esults of size-exclusion c hr omatogr a phy of purified CIB12 and CIB13 are suggesting that both proteins exist as a dimer in solution (75.9 kDa and 78.3 kDa with predicted monomeric masses 56.8 kDa and 56.1 kDa, r espectiv el y).The formation of protein dimers for CIB12 and CIB13 was also verified using the quaternary structure prediction server PDBePISA (buried areas 1117 Å 2 and 1440 Å 2 , r espectiv el y).A Dali searc h for structur all y homologous pr oteins in the PDB identified thr ee GH1 structures with the β-glucosidase TsBGL from Thermofilum sp.ex4484_79 (PDB code 7F1N; Chen et al. 2021 ) as the best match for both CIB12 and CIB13 (Z-score 51.3 and 51.5, root mean square deviation 1.6 Å and 1.8 Å , sequence identity 40% and 41%, respectiv el y).The other top hits include two GH1 β-glucosidases: the thermostable Bgl1317 from a soil metagenome (PDB code 6IER, 28% sequence identity to CIB12 and CIB13) and aku BGL from a sea slug (PDB code 8IN1, 24%-25% sequence identity) (Liu et al. 2019 ).
The crystal structures of CIB12 and CIB13 also r e v ealed their active sites with two catalytic glutamates located inside of the TIM barrel near its C-terminal side (Fig. 7 ).In the retaining catal ytic mec hanism of GH1 hydr olases, one of the two glutamates acts as an acid/base catalyst (Glu204 in CIB12 and CIB13) and the other acts as a nucleophile (Glu388 in CIB12 and Glu385 in CIB13) (Ketudat Cairns and Esen 2010 ).T he clea v a ge of the glycosidic bond involves the formation of a covalent glucosylenzyme intermediate, which is then deglycosylated in a hydrol-ysis reaction via the attack of a water molecule producing the free enzyme and glucose.Both active sites show the presence of an additional area of electron density in each protomer located near the catalytic glutamates: Glu204 (3.9 Å ) and Glu388 (2.8 Å ) in CIB12 and Glu204 (3.5 Å ) and Glu385 (2.8 Å ) in CIB13 (Fig. 7 , Fig. S5 ).In CIB12, this density was inter pr eted as gl ycerol (two molecules in the CIB12 dimer), whereas the CIB13 dimer contained one molecule of gl ycer ol in one protomer and one molecule of tris(h ydroxymeth yl)aminomethane (Tris) in the second protomer (Fig. 7 ).The glycerol molecule in the CIB12 active site was also positioned close to the side chains of conserved His148 (3.1 Å ), Trp149 (4.5 Å ), Tyr321 (3.9 Å ), Trp426 (4.1 Å ), Glu433 (2.9 Å ), and Trp434 (3.4 Å ).Similarly, the Tris molecule in CIB13 was located near His148 (3.2 Å ), Trp149 (3.7 Å ), Tyr320 (2.6 Å ), Trp423 (3.2 Å ), Glu430 (3.4 Å ), and Trp431 (3.2 Å ).Additionall y, both activ e sites contained se v er al ar omatic and c har ged r esidues, whic h might contribute to substrate binding including Gln16, Trp30, Asn203, Glu207, Tyr341, Trp362, and Phe442 in CIB12 and Gln16, Trp30, Asn203, Phe340, Met357, and Trp359 in CIB13.The crystal structure of CIB12 also r e v ealed the pr esence of the disulfide bond Cys172-Cys212 located near the activ e site, whic h is not conserv ed in CIB13 and might contribute to a higher thermotolerance of CIB12 activity compared to CIB13 (Fig. 4 ).Surface c har ge anal ysis of both pr oteins r e v ealed the presence of several positively or negatively charged patches with primaril y negativ el y c har ged surfaces near the substr ate-binding pocket ( Fig. S6 ).Furthermore, analysis of surface hydrophobicity demonstrated the predominance of polar residues in both proteins with a few hydrophobic patches including the active site opening (e.g.T rp30/T rp30, T yr321/T yr320, T yr341/Phe340, and T rp434/T rp431), which might be involved in cellulose binding ( Fig. S6 ).Site-directed mutagenesis (alanine replacement) of CIB12 and CIB13 confirmed the important role of both catalytic glutamates (Glu204/Glu388 and Glu204/Glu385, r espectiv el y) and several other active site residues (His148 and Trp426 in CIB12, Tyr320 and Trp431 in CIB13) for enzymatic activity of both proteins, whereas the Asn203Ala and Phe340Ala mutant proteins retained detectable activity ( Fig. S7 ).

Physiological and ecological roles of β-glucosidases: growth of C. divulgatum S5 is enhanced by cellobiose
Our work on c har acterization of GH1 enzymes, and their ability to hydr ol yse cellobiose and lactose pointed at their potential function in vivo , namely utilization of these compounds for gr owth.Cultiv ation of C. divulgatum S5 with 3 mM (or 0.1%) cellobiose and lactose in the presence of beef extract (0.3%) revealed significant, up to 50%, increase in biomass yields with cellobiose (Fig. 8 ), but not lactose.Cellobiose was indeed consumed during the growth, with its residual concentration in the medium being ∼25% at the end of the experiment.No growth of the strain was observed with either disaccharide alone , i.e .without beef extract, while concentrations of these disaccharides remained at the initial le v els.Sim ultaneousl y, no measur able le vels of glucose were detected in HPLC chromatograms of supernatants of cellobiose-spiked cultures, suggesting its hydr ol ysis a ppears inside the cells, consistently with the predicted intracellular localization of tested glycosidases and with our pr e vious study that sho w ed no gro wth stimulation b y glucose (Golyshina et al. 2016b ).In the C. divulgatum S5 genome, both CIB12 and CIB13 do not appear to be associated with any operon and are encoded by single genes without signal peptides suggesting that they are intracellular proteins (Golyshina et al. 2016b ).From the proteomic data obtained earlier (Bargiela et al. 2020 ), both proteins were detectable, albeit at low basal le v els, corr espondingl y, 0.017% and 0.014% of the total proteome under standard cultivation conditions , i.e .without addition of gl ycosidic substr ates.In a broader context, all Thermoplasmatales cultured so far, do rely on pe ptide-, or oligope ptide substr ates-based diets.Yeast extr act, tryptone, and/or beef extract are hence essential ingredients in their media, ho w e v er some thermophilic members of this order produce higher biomass yields in presence of mono-, or disaccharides (Huber and Stetter 2006 ).The apparent ability to utilize intermediates of the breakdown of cellulosic compounds, previously not reported in Cuniculiplasma spp., points at their extended potential to colonize niches where they are neighbouring the primary (pol ysacc haride) pr oducing or ganisms , e .g. Dunaliella sp., C. acidophila , E. mutabilis , and Bryopsida sp.(Distaso et al. 2022 ).In photoheter otr ophs, the ability to degrade cellulose by secreted endoβ-1,4-glucanases to cellobiose, with its consequent transport and assimilation was earlier demonstrated for Chlamydomonas spp.(Blifernez-Klassen et al. 2012 ).The ability to hydr ol yse cellulose is also known for heter otr ophic acidophilic/acidotoler ant bacteria, for example, in Acidisoma spp.from Rhodospirillales (Mieszkin et al. 2021 ), Alicyclobacillus spp .(K usube et al. 2014 ), Acidothermus spp.(Mohagheghi et al. 1986 ), and acidobacteria (Kielak et al. 2016, Gonzàlez et al. 2020 ), to name just a few examples .T his points at the intrinsic microbial enzymatic potential to degrade cellulosic polymers in acidic systems to provide oligosaccharides, including cellobiose, to noncellulolytic organisms .T hus , Cuniculiplasma spp., and likely, other Thermoplasmatales as well, may become a part of a wider cellulose-degrading community and significantly contribute to the carbon cycling thanks to their intracellular cellobiohydrolases.

Conclusion
In this w ork, tw o r epr esentativ es of gl ycosidases of GH1 famil y from C. divulgatum were characterized.Both CIB12 and CIB13 were activ e a gainst cellobiose and lactose but sho w ed substr ate pr eference to cellobiose, and ther efor e wer e identified as cellobiohydrolases, EC 3.2.1.21.Both enzymes exhibited activities at broad pH r anges with slightl y acidic optima and at temper atur es as high as 60 • C, with the optimum at 50 • C. We suggest the robust glycosyl hydrolase activities of these enzymes at elevated temperatures ma y ha v e been inherited fr om their thermophilic ancestors, and have been retained even after the colonization of colder environments .Furthermore , their ability to hydrolyse the cellobiose, the intermediate product of degradation of cellulosic materials is a nov el tr ait identified in C. divulgatum and in acidophilic arc haea inhabiting acid mine dr aina ge sites .T his trait expands the range of substrates used by C. divulgatum , which was assumed to be limited to oligo-and polypeptides .T his also implies an increased scale of their involvement into the carbon cycling, suggesting greater ecological significance of these archaea in acidic en vironments .

Figure 2 .
Figure 2. GH activity assays of CIB12 (A) and CIB13 (B) for against 21 chromogenic GH substrates.CIB12 and CIB13 (3 μg for each) were incubated with indicated p NP-substrates (1 mM each) at 37 • C for 2 h, and the released reaction product p -nitrophenol was measured at 410 nm.

Figure 3 .
Figure 3. Hydr ol ytic activity of CIB12 (A) and CIB13 (B) against natural GH substrates.Purified CIB12 and CIB13 (3 μg each) were incubated with 1 mM substr ates ov ernight at 30 • C, and 10 μl aliquots of r eaction mixtur e wer e used for the anal ysis of pr oduced r educing sugars using a modified BCA assa y.T he results are means ± SD from at least two independent experiments.

Figure 4 .
Figure 4. GH activity of purified CIB12 and CIB13 as a function of pH (A and B), temper atur e (C and D), NaCl concentration (E and F), and Tween-20 concentration (G and H).GH activity of proteins was determined using 2 mM p NP-ß-d -glucopyranoside (A, B, C, D, G, H: 3 μg of protein/reaction, 2 h incubations) or 25 mM cellobiose (E, F: 5 μg of pr otein/r eaction, 4 h incubations) as substrates in 50 mM MES buffer (pH 5.0) at 30 • C (or as indicated on gr a phs).

Figur e 5 .
Figur e 5. T hermostability of CIB12 and CIB13: activity-based (A and B) and fluorescence (C and D) analyses.(A and B) Purified proteins were incubated at indicated temper atur es (30 • C-80 • C) for differ ent time (1-5 h), and the r emaining enzyme activity was measur ed at 30 • C using 2 mM p NP-βd -glucopyranoside as substrate.(C and D), Determination of protein melting point ( T m) of enzymes by differential scanning fluorescence (DSF) using SYPRO Orange dye.Temper atur e-induced pr otein unfolding was monitor ed in r eal-time using 10 μg of pr otein/sample (0.2 ml).

Figure 6 .
Figure 6.Crystal structures of CIB12 and CIB13: ov er all fold of protomers related by 90 • rotations .T he proteins are shown as ribbon diagrams with the active site glutamates shown as sticks.

Figure 7 .
Figure 7. Crystal structures of CIB12 and CIB13: close-up view of the active sites.(A) CIB12 and (B) CIB13.The proteins are shown as ribbon diagrams with the side chains of active site residues shown as sticks including catalytic glutamates (E204 and E388 for CIB12 and E204 and E385 for CIB13).The bound ligand molecules are shown as sticks and labelled as GOL (glycerol) and TRIS (tris(h ydroxymeth yl)aminomethane).

F igure 8 .
Gro wth of C. divulgatum cultures with beef extract and consumption of added disaccharides.(A and B), Growth of C. divulgatum cultures (OD, 600 nm) in DSMZ medium 88 (see the section "Materials and methods") with/without addition of beef extract (0.3% w/v), cellobiose (0.1% (w/v), and/or lactose (0.1%).(C and D), Residual concentrations of cellobiose and lactose during growth of C. divulgatum cultures, as revealed by HPLC analysis of culture supernatants.